The field of this invention is prodrug activation. More particularly, the present invention pertains to a compound that includes an active therapeutic agent attached to a blocking group, methods for making such compounds and methods of converting such compounds to active therapeutic agents using antibodies having aldolase activity.
Drug therapy can be limited by nonspecific toxicity. To overcome this limitation, several approaches towards a site-selective therapy have been suggested. Selective therapy can be based on the enzymatic activation of a prodrug at a target site. Unless the target displays a specific enzymatic activity that can be used for prodrug activation (Denmeade et al., Cancer Res 58, 2537-2540, 1998), the enzymatic activity has to be conjugated to an antibody that binds to a target cell surface antigen selectively expressed at the target site. The antibody-enzyme conjugate is injected first. Once it has accumulated at the site and has been cleared from the periphery, the prodrug is administered. The prodrug is selectively activated by the targeted enzymatic activity. One molecule of enzyme catalyzes the activation of many molecules of prodrug. This inherent amplification feature of the system allows the generation of high drug concentrations at the target site. The concept of antibody-directed enzyme prodrug therapy, termed ADEPT, has been developed by Bagshawe, Senter, and others (Bagshawe et al., Br. J. Cancer 58, 700-703, 1988; Senter et al., Proc. Natl. Acad. Sci. USA 85, 4842-4846, 1988; Niculescu-Duvaz, et al., Adv. Drug Delivery Rev. 26,151-172, 1997). A number of antigens that are expressed on the surface of cells have been shown to be effective targets for antibody-mediated therapy. Thus, the antibody component is not the critical parameter for ADEPT. By contrast, the requirements for the enzyme component for ADEPT are difficult to achieve. First of all, selective prodrug activation requires the catalysis of a reaction that must not be accomplished by endogenous enzymes in blood and normal tissue of the patient. Enzymes of non-mammalian origin that meet these needs are, however, likely to be highly immunogenic, a fact that makes repeated administration impossible. There is a need in the art, therefore, for improved ADEPT compounds and methods.
In one aspect, the present invention provides a compound according to formula I, below 
In formula I, X is a heteroatom of a target molecule and Y is absent or a self-immolative linker such as shown below: 
Each R is independently hydrogen, C1-C6 alkyl, C1-C6 alkenyl, C5-C6 aryl or a heterocycle containing five or six ring atoms. In one embodiment, the heteroatom is a nitrogen, oxygen or sulfur atom in a functional group of the target molecule. Preferred target molecules are therapeutic agents or fluorescent molecules. Exemplary and preferred therapeutic agents include anti-tumor agents such as a cytotoxic agent, a microtubule stabilizing agent or an antibiotic. A preferred antibiotic is an anthracycline antibiotic such as doxorubicin or a therapeutically active analog thereof. A preferred microtubule stabilizing agent is paclitaxel, epothilone, or a therapeutically active analog thereof.
In another aspect, a compound of the present invention includes a compound having the structure II, below 
where X, Y and R are as defined in reference to formula I.
An especially preferred compound of this invention has the structure 
where R9 is CH3, CH2F, CH2Cl, CH2CH3, CH2OOCCH3 or CHxe2x95x90CH2 and each B is independently H, 
where Y and R are as defined above and with the proviso that two Bs are H.
In another aspect, this invention provides a of converting an inactive molecule to an active molecule. The process includes the step of exposing the inactive molecule to an agent that catalyzes a retro-Michael reaction. A preferred inactive molecule is a compound according to formula I or II, above. A preferred agent that catalyzes a retro-Michael reaction is a protein. A preferred is an antibody, the catalytic activity of which is inhibited by a xcex2-diketone compound. Exemplary and preferred such antibodies are 38C2 or 33F12. The process can occur in vitro, in situ or in vivo. In one embodiment, the antibody is a bifunctional antibody that specifically immunoreacts with a cell surface antigen of a target cell such as a tumor cell or a virus-infected cell. The antibody can be a single chain antibody.